Translocation Studies Mid-Term Review (MTR) Meeting Marseille, France F. Vidal-Aroca, M.G.P. Page and J. Dreier Marie Curie Actions Research Training Networks (RTN)

Deteriorating situation regarding treatment of Gram-negative infections Growing concern over: Multi-resistant Acinetobacter Multi-resistant Pseudomonas aeruginosa Carbapenem-resistant Klebsiella pneumoniae ESBL-producing K. pneumoniae and Escherichia coli Warnings for: Carbapenem-resistant Enterobacter GNNFs like Burkholderia, Stenotrophomonas…. Background

Interests & Expectations at Basilea Insight into the permeation of  -lactam antibiotics in clinical isolates Permeation route(s) of novel compounds Role of porins in resistance Role of efflux systems in resistance

Rationale Antibiotic Uptake Efflux Antibiotic-resistant Cells Focus on the role of porins and efflux systems in antibiotic resistance of Gram-negative bacteria

Bridged monobactams are potent  - lactamase inhibitors When R2 = H, activity against  - lactamase in situ can be modulated by R1 When R2 = OMe or larger, all activity against  -lactamase in situ is lost, irrespective of R1 Permeability of Bridged Monobactams

R2 IC 50  - lactamase in vitro (mM) MIC against cells in situ Effect on OMPF Conductance (from Mathias) RO OCH >64*No interaction RO CH.CONH >64*Blocking RO Cl 0.1>64*No interaction RO S-tetrazole 0.009>64*Blocking RO H 0.1 2*No effect Carbenicillin - - 4No interaction Penicillin G - -16Some blocking * In combination with penicillin

Permeability of Bridged Monobactams Is the lack of correlation due to OmpC?  Knock-out strain with only OmpF Need more data points to look for trends  Select more penicillins with a spread of MIC values  Select more bridged monobactams Are the conditions appropriate?  low pH, high salt vs neutral pH, isotonic

Gene Disruption Baba T. et al. Molecular Systems Biology (2006) Gene target: ompC tolC

Meropenem Aztreonam Cefepime Penicillin G Ampicillin Carbenicllin  -Lactams Selected

E. coli MIC Determination (I)  tolC: MIC determination is ongoing

Conclusions 1. Equal contribution of OmpC and OmpF to aztreonam, ampicillin and carbenicillin susceptibilities. 2. Strong influence of the double K.O. mutant on cefepime and meropenem susceptibilities. 3. Weak influence of the single K.O. mutants on cefepime and meropenem susceptibilities. 4. High osmolarity potentiates the effect of cefepime > penicillin G > others.

Additional Antibiotic Classes Selected Ciprofloxacin GentamicinMinocycline Chloramphenicol Erythromycin Polymyxin B

E. coli MIC Determination (II)

Conclusions 1.The susceptibility of E. coli to gentamicin or polymyxin B is NOT influenced by OmpF and OmpC porins. 2.Minocycline susceptibility is increased in the double K.O. mutant. 3.Deletion of OmpF slightly decreases the ciprofloxacin susceptibility. 4.Chloramphenicol and erythromycin data need to be confirmed

 -Lactamase Inhibitors (BLI) Approach Strains with AmpC overexpressed MICs  -lactamsMICs  -lactams+BLI IC 50 of  -lactamase in situ AmpC ContributionAmpC InhibitionRole of outer membrane & porins SUSCEPTIBILITYSYNERGY PERMEABILITY OF BLI

BLI:  -Lactam Selection Overexpression of AmpC in wt and porin mutant strains MIC determination led to the selection of the following  -lactams antibiotics for synergy test: aztreonam, penicillin G, ampicillin and carbenicillin The bacteria growth was not affected at concentrations of 256 mg/l of bridged monobactans tested The overexpression of AmpC does not influence the MIC of other classes of antibiotics.

BLI: Experiments ongoing Synergy assay I) II) Permeability assay

Pseudomonas aeruginosa Ubiquitous environmental bacterium One of the top causes of opportunistic human infections bacteraemia in burn victims urinary-tract infections hospital-acquired pneumonia AIDS population predominant cause of morbidity and mortality of cystic fibrosis patients Intrinsic resistance to antibiotics and disinfectants

Piddock L.J.V. Clin Microbiol Rev, Apr. 2006, p. 382–402 Efflux Pumps Resistance Mechanism 1- Enzymatic inactivation 2- Alternative metabolic pathways 3- Reduced uptake 4- Alteration of the target site 5- Membrane bound efflux pumps

Efflux Systems in P. aeruginosa SystemSubstrates MexAB/ OprM *  -lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones, sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan,… MexXY/ OprM* aminoglycosides,  -lactams, erythromycin, fluoroquinolones, tetracyclines,… MexCD/ OprJ*  -lactams, quinolones, chloramphenicol, novobiocin, sulfonamides, tetracyclines, trimethoprim, triclosan,… MexEF/ OprN*fluoroquinolones, tetracycline, chloramphenicol, trimethoprim, triclosan… MexJK/ OprMciprofloxacin, erythromycin, tetracycline, triclosan Mex(G)HI/ OpmDvanadium, norfloxacin,… MexVW/ OprMchloramphenicol, erythromycin, fluoroquinolones, tetracycline,… Other clinically relevant bacteria with RND efflux systems: A. baumannii, B. cepacia, B. pseudomallei, S. maltophilia, N. gonorrhoeae, N. meningitidis, S. marcescens, E. coli, S. enterica, E. aerogenes, K. pneumoniae, K. oxytoca, C. jejuni, P. mirabilis, H. influenzae. 7 of 12 RND-pump encoding operons in P. aeruginosa have been characterized: * major contributors to MDR

Knock-out Mutants 1. Make single and multiple knock-out mutants of all major RND pumps in P. aeruginosa. 2. Measure susceptibility to all available antibiotics in comparison to the parent strain. Schweizer H.P. (2003) Genetics and Molecular Research 2(1):48-62

Target gene A B B C 1234 genomic DNA PCR 1/2 & 3/4 Cloning vector Gene replacement vector (Gm R, SacB) 1 st crossing- over: antibiotic resistant & sucrose sensitive 2 nd crossing- over: antibiotic sensitive & sucrose resistant Knock-out mutant (or reversal to wt) Disruption of various mex & opr genes

StrainAffected Efflux-pump Systems PAO1Wild type PAO1  mexR MexAB-OprM overexpression (commonly found in clinical isolates) PAO1  oprJ Impaired assembly of MexCD-OprJ P. aeruginosa Strains Studied

P. aeruginosa MIC Determination (I)

Conclusions 1.Wild type efflux-pump expression caused high resistance to ampicillin, carbenicillin and penicillin G. 2.The mexAB-oprM system strongly contributed to resistance towards piperacillin, aztreonam, meropenem and cefepime. 3.No contribution of the main pumps to imipenem resistance was observed. 4.The oprJ K.O. mutant did not cause significant differences in  -lactam susceptibility.

P. aeruginosa MIC Determination (II)

Conclusions 1.Wild type efflux-pump expression caused high resistance to chloramphenicol, erythromycin and minocycline. 2.The mexAB-oprM system strongly contributed to resistance towards ciprofloxacin and moxifloxacin. 3.No contribution of the main pumps to gentamicin and polymyxin B resistance was observed. 4.The oprJ K.O. mutant did not cause significant differences in susceptibility to the tested compounds.

Consortium Interactions Prof. Winterhalter Compounds sent for Conductance Measurements Summer School Participations Mircea Petrescu’s Training Dr. Weingart Strains Exchange Prof. Gameiro Cephalosporins Provided for Measurements Basilea Pharmaceutica Dr. Ceccarelli & Prof. Ruggerone In silico Modeling: Porins & TolC Ph.D. Program Presentation

Italy Dr. Bertoni UK Dr. Camara USA Prof. Schweizer Acknowledgements Basilea Dr. Caspers Basilea Dr. Shapiro All the Consortium People